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Quantitative Molecular Fluorescence Endoscopy: a new and promising tool to Predict and Evaluate Response to neoadjuvant chemoradiotherapy in Locally Advanced Rectal Cancer patients (#358)
M. Koller1, J. J. J. Tjalma2, M. D. Linssen5, E. Hartmans2, S. J. de Jongh2, A. Jorritsma-Smit5, A. Karrenbeld4, E. G. E. de Vries3, J. Kleibeuker2, J. Pennings6, K. Havenga1, P. H. J. Hemmer1, G. A. P. Hospers3, B. van Etten1, V. Ntziachristos7, G. M. van Dam1, 9, 8, D. Robinson10, W. B. Nagengast2
1 University Medical Center Groningen, Surgery, Groningen, Netherlands
Patients with locally advanced rectal cancer (LARC) are treated by neoadjuvant chemoradiotherapy (nCRT) followed by surgery. To date, there is a growing interest in the non-operative management of patients with a clinically complete response to nCRT, which is associated with good survival rates and reduced long-term morbidity. However, current restaging techniques are suboptimal to identify patients that might benefit from watchful waiting. Therefore, we investigated if quantitative molecular fluorescence endoscopy (Q-MFE) can improve clinical response assessment of nCRT in LARC patients.
We evaluated the feasibility of Q-MFE for response prediction and evaluation with 4.5mg bevacizumab-800CW targeting vascular endothelial growth factor A in 30 patients with LARC. Q-MFE procedures were scheduled at two different time points during the neoadjuvant treatment: the first at baseline prior to the start of nCRT, the second following completion of nCRT. At both time points, fluorescence was visualized using near-infrared molecular fluorescence endoscopy. Additionally, fluorescence signals were quantified in vivo and ex vivo using multi-diameter single fiber reflectance and single fiber fluorescence (MDFSR/SFF) spectroscopy. Results were correlated with current clinical standards: radiological restaging, white-light endoscopy and the pathological outcome of the surgical specimen.
In all procedures performed at baseline, tumor tissue showed clear fluorescence (Q·μfa,x = 3.75·10-4 ) compared to normal rectal tissue (Q·μfa,x = 1.20·10-4). Higher fluorescence signals were seen in tumor tissue (4.62·10-4±0.29·10-4) of good responding patients, whereas patients with lower fluorescence signals in tumor tissue showed an intermediate or poor response to nCRT (3.74·10-4±0.44·10-4 and 2.48·10-4±0.56·10-4). After nCRT, tumor tissue showed significant higher fluorescence compared to normal rectal tissue and fibrosis, with an area under the curve of 0.925. VEGFA-targeted Q-MFE showed a promising correlation to pathological staging of the surgical specimen with for Q-MFE and white-light endoscopy respectively a positive predictive value after nCRT of 92% vs. 90% and negative predictive value of 100% vs. 20%. Overall, Q-MFE correctly changed restaging diagnosis in 4 (16%) of the LARC patients.
VEGFA-targeted Q-MFE showed to be a promising new tool in individualized treatment of LARC patients by prediction of tumor response to nCRT at baseline and recognizing a clinical complete response after nCRT. These results might lead to a paradigm shift in the management of patients with locally advanced rectal cancer.
Quantitative molecular fluorescence endoscopy in rectal cancer patients
A representative example of a patient with locally advanced rectal cancer in which we performed quantitative molecular fluorescence endoscopy. From left to right: A white light image of the tumor with the HD video endoscope, a white light image of the tumor with the fluorescence endoscopy optical fiber, clear fluorescence is seen in the tumor tissue, which was confirmed with histology.
Keywords: molecular fluorescence endoscopy, bevacizumab-800cw, personalised medicine, rectal cancer
In vivo imaging of human monocyte trafficking in a preclinical model of mouse myocardial infarction to study mononuclear cell function in the context of cardiovascular diseases (#92)
J. Iking1, A. Borgscheiper1, L. Honold3, M. T. Kuhlmann3, S. Hermann3, M. Schäfers2, 3, L. Stegger2, E. Pardali1, J. Waltenberger1
1 University Hospital Münster, Dept. of Cardiovascular Medicine, Münster, North Rhine-Westphalia, Germany
Infiltrating monocytes represent a key player in infarct healing and tissue remodelling. We have previously shown that cardiovascular risk factors impede monocyte migratory behaviour in vitro which might also result in higher risk of heart failure after an acute myocardial infarction (MI). So far, however, no in vivo data is available concerning the recruitment of human monocytes into the ischaemic myocardium. Our aim was to study the recruitment and migratory behaviour of human monocytes using a non-invasive in vivo imaging approach in a preclinical model of mouse myocardial infarction.
Human monocytes were labelled with the fluorescent dye DiD and the radioactive tracer 99mTc-HMPAO. Immuno-deficient NOD/Scid mice underwent surgery for permanent ligation (PI) of the LAD coronary artery. Area at risk of infarction (AAR) was determined by myocardial perfusion SPECT/CT subsequently to surgery. 1 or 3 days after surgery, 10x106 dual-labelled human monocytes were injected into the tail vein of operated NOD/Scid mice and in vivo SPECT/CT scans were acquired ~18h p.i. (2 or 4 days post MI). Subsequent to SPECT/CT scans, mice were sacrificed and organs were assessed for biodistribution of human monocytes by fluorescence reflectance imaging (FRI), autoradiography (AR), γ-counting, and histology.
The dual-labelled human monocytes can be detected in vivo in the AAR by SPECT/CT imaging on day 2 and day 4 post MI. Ex vivo SPECT/CT scans of the heart further revealed that the human monocytes mainly accumulate in the border zones of the infarcted area. Quantification of the SPECT data displayed higher signal in the AAR of PI mice when compared to remote myocardium and sham mice on day 2 and day 4 post MI. Similar results were obtained with ex vivo FRI, AR and γ-counting of sham and PI hearts. In addition, the signal intensity of SPECT/CT, FRI and γ-counting positively correlates with the size of the AAR suggesting that numbers of infiltrating monocytes increase with the severity of MI.
Using a complementary spectrum of different techniques, we have established a non-invasive imaging model to visualize and measure the extent of human monocyte trafficking into the ischaemic myocardial tissue. This novel model bears great potential to explore the migratory behaviour and biodistribution of human immune cells isolated from different patient populations (e.g. diabetes, coronary artery disease, post MI) in vivo.
Pardali, E., Schmitz, T., Borgscheiper, A., Iking, J., Stegger, L., & Waltenberger, J. (2016). Cryopreservation of primary human monocytes does not negatively affect their functionality or their ability to be labelled with radionuclides: basis for molecular imaging and cell
This research was funded by DFG, CRC 656 Münster, project C12 and the DSHF, project F732/13.
In vivo & ex vivo detection of human CD14 monocytes after myocardial infarction by SPECT/CT
Fig. 1: Dual-labeled human CD14++ monocytes can be detected in and ex vivo by SPECT/CT in the area at risk of infarction on day 2 and day 4 post MI but not in the hearts of sham operated mice. Ex vivo SPECT/CT scans further reveal a higher signal in the border zones of the infarcted area. The data is only statistically significant if explicitly stated (* p < 0.05, ** p < 0.01).
Keywords: myocardial infarction, FRI, monocytes, migration, SPECT, in vivo
Hybrid label-free microscopy by means of optical-resolution optoacoustic and non-linear optical modalities (#237)
M. Seeger1, 2, V. Ntziachristos1, 2
1 Technical University Munich, Chair for biological Imaging, Munich, Bavaria, Germany
Precise examination of biological moieties, such as vasculature and the connective tissue, and their mutual interaction are favored for achieving new insights into both general knowledge of biological systems as well as pathophysiological hallmarks. For that, high resolution, high penetration depth as well as label-free sensing is desired. In this project, we therefore devised a hybrid microscope based on label-free optoacoustics and non-linear optical modalities achieving resolutions of ~1 µm at depth up to 500 µm and at imaging speeds of up to 1 Hz framerate.
The custom-built hybrid microscope is based on raster-scanning diffraction limited focused optical excitations across a sample by a via a set of galvanometric mirrors. For the optical-resolution optoacoustic subsystem (OR-OAM), a 532 nm laser emitting 1.4 ns pulses running at 50 kHz is utilized to generate optoacoustic signals being detected by a spherically focused piezo-electric transducer above the sample in transmission mode and coupled to the sample by a buffer droplet. The non-linear optical (NLO) modalities, i.e. two-photon excitation fluorescence as well as second and third harmonic generation, are based on a 1043 nm fs-laser being co-aligned with the OR-OAM subsystem. To achieve high-speed scanning, a streaming like acquisition mode is enabled.
The system was characterized to achieve resolutions of ~1 µm laterally and ~5 µm axially for all equipped modalities at a maximum depth of up to 500 µm and to sense label-free a huge variety of biological relevant compounds e.g. vasculature, collagen, elastin, keratinocytes, hair follicle and foam cells. This allowed us to study not only histological tissue samples but also small model organisms. For histopathological examinations, we imaged human carotid atheroma samples and achieved new insights into microscopic interactions among blood embeddings and the connective tissue, namely collagen and elastin. For animal studies, we imaged mouse ears as well as zebrafish larvae, and were able to transfer the compatible examination of the mentioned moieties in vivo. In here, we yielded single-cell resolution allowing investigations of the vasculature system as well as comprehensive depiction of the microstructural composition.
In this work, we showcased a novel approach for imaging biological samples starting from single cells over tissue slices up to small model organisms with cellular resolution, label-free contrast mechanisms at speed and penetration depth required for extensive examination of biological and biomedical processes on the microscale. This allowed us to not only enlarge the knowledge of the composition and structure of biological tissue but also gave new insights into mutual interaction among the most important moieties and its subsequent potential role in pathophysiological diagnosis.
Schematic depiction of the hybrid microscopy system
Abbreviations: AMP, amplifier; DAQ, data acquisition card; F, optical filter; FM, flippable mirror; GM, galvanometric mirrors; GMC, GM control; L, lens; LP-DM, longpass dichroic mirror; M, mirror; ND, neutral density filter; OL, microscope objective lens; P, prism; PMT, photomultiplier tube; S, xyz stage; SP-DM, shortpass dichroic mirror; UT, ultrasound transducer
Label-free multimodal microscopy of an in vivo mouse ear
Microscopic examination of the vasculature system by optical-resolution optoacoustic microscopy (OR-OAM), hair follicle by two-photon excitation fluoresence (TPEF), extracellular collagen network by second harmonic generation (SHG), and the overall tissue morphology and keratynocytes by third harmonic generation (THG).
Keywords: Optoacoustic, Photoacoustic, multimodal, microscopy, label-free